This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. A.Specific Aims The ability of the intestinal ameba Entamoeba histolytica to phagocytose host cells correlates with parasite virulence, but the mechanisms underlying this process and its specific contribution to pathogenesis remain unknown. The specific aims were not changed during the last year. Aim 1: Test the hypothesis that E. histolytica has a phagocytosis receptor specific for the collagenous tail of C1q and the collectins. Aim 2: Test the hypothesis that the serine-rich E. histolytica protein (SREHP) and/or the related asparagine-rich Ariel proteins function as receptors for phagocytosis of killed cells. B.Studies and Results Aim 1: As noted last year, we discovered that E. histolytica phagocytoses apoptotic cells coated with C1q more efficiently than apoptotic cells coated with control proteins. To follow up on this, we developed a method to construct "single-ligand" fluorescent particles by biotinylating protein ligands of interest and using them to coat streptavidin-latex beads. With this system, we demonstrated that C1q alone is a potent stimulant of E. histolytica phagocytosis. Mannose binding lectin (MBL), a collectin family member that is structurally related to C1q by virtue of a common collagen-like tail domain, also stimulated E. histolytica phagocytosis, as did purified collagenous tails from C1q and surfactant protein A (SP-A). Entamoeba histolytica also migrated towards these proteins in transwell assays. These findings were published last October. We have now used flow cytometry to assay binding of FITC-labeled collectins to the surface of E. histolytica. The results showed that C1q-FITC binds to the amebic surface in a saturable manner, and that binding of C1q-FITC can be partially competed with free MBL. These data provide additional evidence of an amebic receptor specific for the collagenous collectin tail. We are actively engaged in identifying this receptor. Our first approach was to use purified C1q as the bait in cross-linking experiments using the multifunctional cross-linker Sulfo-SBED, which transfers biotin to proteins in close proximity. Our initial experiments were unsuccessful, because the C1q bait forms multimers resulting in overwhelming biotinylation of other bait molecules. A similar approach using purified collagenous tails might work, as might affinity-based methods. An additional approach we have taken is to use flow cytometry to sort amebic trophozoites that are positive or negative for phagocytosis of C1q-coated beads. We have compared the membrane protein profiles of trophozoites positive for phagocytosis of C1q-coated beads to the membrane protein profiles from non-phagocytic trophozoites and those that phagocytosed BSA-coated control beads. Enrichment of specific membrane proteins is observable on silver-stained SDS-PAGE gels. After optimizing our FACS sorting protocol further, we will identify the reproducible bands using mass spectrometry. Although we found that collagenous tails from SP-A significantly stimulate E. histolytica phagocytosis, intact SP-A actually inhibited phagocytosis. Phagocytosis of SP-A-coated particles was approximately five times lower than basal phagocytosis of beads coated with bovine serum albumin (BSA, negative control). This raises the possibility that the lectin domain of SP-A binds to an inhibitory receptor on the ameba surface, while the SP-A tail stimulates phagocytosis via a common collectin receptor. Consistent with this possibility, SP-A can inhibit or enhance macrophage phagocytosis based on context. That is, in the absence of bacteria or cellular debris, SP-A binds an inhibitory receptor via its lectin domain, but in the presence of bacteria or cellular debris it preferentially binds the debris, which exposes its collagenous tail and stimulates phagocytosis. Our ability to pursue this possibility further has been limited by inadequate quantities of SP-A. Thus, we have developed a method to isolate SP-A from a human lung carcinoma cell line, which we are in the process of optimizing. Once we have adequate amounts of SP-A, we plan to confirm its biological activity with bacterial binding studies and then to use it to further examine if SP-A may have the ability to either stimulate or inhibit E. histolytica phagocytosis depending on the local environment. Aim 2: Some of our results from aim 2 were published in 2008 in a paper which describes a monoclonal antibody screen we conducted to identify phagocytosis inhibitory antibodies. In this screen, we identified the SREHP as the target of an inhibitory antibody. In follow-up work, we found that the Fab fragment of the inhibitory antibody had no effect on phagocytosis and that recombinant SREHP produced in Escherichia coli had no specific binding activity. The SREHP is a glycoscylated protein, so we want to express it on the surface of Chinese Hamster Ovary Cells and conduct binding studies. The gene has been cloned into the vector pDisplay (which incorporates a mammalian signal peptide and membrane anchor) for this purpose, but the experiment has not yet been completed. In addition, we want to silence expression of the SREHP in amebic trophozoites. We have constructed four plasmids for expression of small hairpin RNAs based on four different SREHP sequences (and a scrambled control vector), but we have not been able to transfect E. histolytica successfully with these constructs. C. Significance: Entamoeba histolytica's phagocytic ability correlates with virulence and these studies promise to clarify its underlying mechanisms. Aim 1 is focused on clarifying the ligands on apoptotic cells that trigger E. histolytica phagocytosis and on the amebic receptors that bind them. These receptors may be candidates for inclusion in a vaccine to prevent amebiasis. Furthermore, if we confirm that SP-A inhibits or enhances amebic phagocytosis depending on context, then it may suggest a completely novel mechanism by which E. histolytica senses its environment and this could provide critical insights into why E. histolytica is invasive in only about 10% of infections. Successful silencing of the SREHP in aim 2 would extend results of our antibody study, and demonstration of binding of apoptotic cells and/or bacteria to CHO cells expressing the SREHP would definitively demonstrate that this protein functions as an adhesin. Though the SREHP is a leading vaccine candidate, its function is unknown;therefore, this would be very significant. D. Plans: An R01 grant on the phagocytosis studies in aim 1 was funded by the NIAID in 9/2008. Accordingly, the specific aims will be changed entirely in the coming year if I am allowed to remain on the COBRE grant. The new aims will focus on an E. histolytica homologue to leishmanolysin. Leishmanolysin is an immunodominant surface protein of leishmania promastigotes, and is an M8-type metalloproteinase that degrades complement and immunoglobulins. Interestingly, the Drosophila orthologue invadolysin plays critical roles in cell division and cell migration. We identified two leishmanolysin homologues in the E. histolytica genome, which we named E. histolytica leishmanolysin-like proteins 1 and 2 (EhLMLP-1 and -2). The EhLMLP-1 gene is not present in Entamoeba dispar, a non-pathogenic ameba that is closely related to E. histolytica. Few differences in the protein coding regions have been identified between E. histolytica and E. dispar. This and the importance of leishmanolysin in leishmania virulence make it important to determine the function of EhLMLP-1 and the specific role(s) it plays in E. histolytica biology. We plan to: 1) determine if EhLMLP-1 is a cell surface metalloproteinase;2) determine if EhLMLP-1 contributes to immune resistance by degrading IgG and converting complement C3b to iC3b;and 3) determine if EhLMLP-1 and -2 function in cell division and migration. E. Publications (since the 2008 progress report) Teixeira JE, Heron B, Huston CD. C1q- and collectin-dependent phagocytosis of apoptotic host cells by the intestinal protozoan Entamoeba histolytica. Journal of Infectious Diseases. 2008. 198:1062-1070. Vaithilingam A, Teixeira JE, Huston CD. Addenda article: Endoplasmic reticulum continuity in the protozoan parasite Entamoeba histolytica: evolutionary implications and a cautionary note. Communicative and Integrative Biology. 2008. 1(2):172-174. Mentoring Summaries: Dr. Markus Thali Dr. Thali meets with Dr. Huston on a monthly basis to discuss research and personnel management issues. He also served on the thesis committee of one of Dr. Huston's graduate student (Brad Heron) and, as the director of the Cell and Molecular Biology Graduate Program, he is somewhat familiar with the progress of the other graduate student in Dr. Huston's lab (Archana Vaithilingam). In addition, since fall 2007, the Huston and the Thali groups meet once a month for a joint group meeting. Dr. Cory Teuscher Chris previously had issues with a graduate student that he successfully resolved. We discussed on several occasions his interests and desire to submit a second R01 application. We talked in depth about the two fundamental approaches to successfully running an academic research enterprise: highly focused with sustained productivity in a single area/molecule/pathway vs. broad based with multiple mutually non-exclusive areas of interests and expertise. The strengths and weaknesses of each approach were discussed, particularly from the perspective of a junior faculty member with clinical responsibilities. Dr. Gary Ward Dr. Ward meets biannually one-on-one with Dr. Huston to discuss data and the overall direction of Dr. Huston's work, and more frequently on an informal basis when issues related research or personnel management arise. Dr. Ward critically reads and provides feedback to Dr. Huston on his manuscripts and grant applications, and serves on the dissertation committee of one of Dr. Huston's Master's students, Brad Heron. Dr. Ward and Dr. Huston participate in a joint, biweekly lab meeting, which also includes one of the other junior investigators on the COBRE grant, Dr. Matrajt. This data-centered meeting is highly interactive, and an excellent way for Dr. Huston and his students and postdocs to receive regular feedback on the course of their research. Dr. Ward has made an effort to introduce Dr. Huston to - and facilitate his interactions with - others on campus or in the immediate area that might be helpful to him in his research. For example, Dr. Ward recently nominated Dr. Huston to speak at the annual Dartmouth College Molecular Pathogenesis retreat;his talk was very well received, and he now has a number of new contacts in the Dept of Microbiology and Immunology at Dartmouth Medical School. Dr. Ward also provides every opportunity to Dr. Huston to meet with visiting scientists and seminar speakers who come to UVM.